Finding of induced pluripotent stem cells (herein, often referred to as “iPS cells”) (Non Patent Literatures 1 to 3) has increased the momentum for practical use of regenerative medicine using it. The iPS cells are pluripotent stem cells capable of differentiating into various tissues and organs, as with embryonic stem cells (ES cells), but has many advantages over the embryonic stem cells. For example, embryonic stem cells are established from a fertilized egg and thus involve ethical problems, and in contrast, iPS cells can be established from somatic cells and thus do not develop the ethical problem. In addition, embryonic stem cells often cause rejection after transplantation due to difference of a major histocompatibility antigen (MHC), and in contrast, iPS cells are established from cells from a subject for transplantation and thus rarely cause rejection.
On the other hand, the number of iPS cells in the order of 106, a quantity commonly used in a laboratory, is far from sufficient for using iPS cells in regenerative medicine or the like, and required to be in the order of 109 to 1010 for clinical use. However, techniques for mass culture of iPS cells have not been fully established. To culture iPS cells while they are maintained in an undifferentiated state, it is generally considered to be necessary to culture them on feeder cells such as primary cultures of mouse embryonic fibroblast (MEF) and STO cells. However, contamination with a feeder cell constitutes a significant obstacle to use of iPS cells for regenerative medicine. In view of this, studies on a feeder-free cell culture method have been also conducted, and methods enabling cell culture without any feeder cell have been developed, such as a method of culturing iPS cells on the surface of a base material coated with Matrigel and a culture method utilizing a coating with laminin or a partial peptide of laminin. In addition, bag culture has been performed in place of common dish culture. However, it is necessary to repeat culture on a base material with a coating even in a feeder-free culture system, and thus the culture process is complicated and the cost of culture significantly increases, which causes a serious problem of huge cost to treat one patient. A method for efficiently mass-culturing stable iPS cells retained in an undifferentiated state has not been developed yet.
To construct a three-dimensional tissue from cells, it is typically needed to perform three-dimensional culture with an appropriate scaffold material, or to perform spinner culture. However, conventional spinner culture applies a strong mechanical stimulus and significant damage to cells, and thus it is difficult to obtain a large tissue, and even if a large tissue is obtained, the inside often undergoes necrosis. As a countermeasure against this, there exists a series of bioreactors designed to optimize the weight. An RWV (Rotating Wall Vessel) bioreactor, one of such bioreactors, is a rotary bioreactor with gas exchange function developed by NASA. The present inventors have conducted research and development of, for example, a technique of cartilage regeneration from bone marrow cells, etc., by three-dimensional culture with the RWV bioreactor (Patent Literatures 1 to 3).
In contrast, a method for three-dimensionally culturing iPS cells, growth of which in an undifferentiated state is considered to be difficult in an environment without scaffolds such as feeder cells or a coating material, in an efficient manner has not been developed yet.